Macrophages are immune cells that rapidly migrate to sites of infection or tissue damage. Depending on chemical cues in the environment, they become activated to either pro-inflammatory or immunosuppressive phenotypes and subsequently regulate the immune response. Macrophages are therefore critical for maintaining tissue homeostasis, and their improper behavior triggers various diseases. For example, accumulation of pro-inflammatory macrophages in tissues causes chronic inflammation and can lead to atherosclerosis, obesity, and diabetes. On the other hand, immunosuppressive macrophages are recruited to tumors, where they stimulate new blood vessel formation and promote metastasis by migrating ahead of cancer cells. Thus the ability to control the migration of specific types of macrophages in vivo could serve as a useful therapeutic intervention. Recently, studies have observed that pro-inflammatory macrophages are less motile than unactivated macrophages in vitro, which could potentially explain why they accumulate in tissues during inflammatory diseases. The goal of this proposal is to explain why macrophages have reduced motility after exposure to pro-inflammatory cues and to identify the specific molecules that are responsible for this behavior. Two hypotheses will be tested in vitro using human macrophages that are isolated from blood and activated to a pro-inflammatory phenotype. First, pro-inflammatory macrophages could be less motile because they have lower expression of two chemokine receptors: CXCR4 and CSF1R, which mediate the migration of unactivated macrophages towards the chemokines CXCL12 and M-CSF1. This hypothesis will be tested in Aim 1 by genetically engineering pro-inflammatory macrophages to overexpress CXCR4 or CSF1R and determining whether their motility towards their ligands is rescued. Second, since pro-inflammatory macrophages highly overexpress both the chemokine receptor CCR7 and one of its ligands CCL19, the cells could have reduced motility towards CCR7 chemokines because CCL19 overwhelms and desensitizes the receptor. This hypothesis will be tested in Aim 2 by reducing CCL19 expression in pro-inflammatory macrophages with short hairpin RNA and determining whether this enables them to migrate towards gradients of CCL19 and CCL21, which is the other CCR7 ligand. In addition, since macrophages have been shown to switch between different activation phenotypes in vivo, we hypothesize that reprogramming pro-inflammatory macrophages to an immunosuppressive phenotype will directly enhance their motility. This hypothesis will be tested in Aim 3 by treating pro-inflammatory macrophages with the immunosuppressive cytokine IL-4 and measuring changes in migration towards CXCL12, M-CSF1, and other chemokines. These studies will help identify the molecules responsible for reducing the motility of macrophages after pro-inflammatory activation, which could potentially lead to novel therapies for forcing pro-inflammatory macrophages to migrate out of inflamed tissue.